The V0 sector subunit "a" has been shown to play important roles in diverse membrane fusion or secretion functions independent of the V-ATPase proton-pumping activity. These roles include vacuolar fusion, synaptic vesicle exocytosis of neurons, exocytosis in apical secretion of exosomes and morphogens, exocytosis of insulin secretion from pancreatic islets and phagosomal fusion in microglial-mediated neuronal degradation. The molecular mechanism and regulation of all these secretion/membrane fusion events are largely unknown. The neuronal subunit a1 (V100 in fly) is required for SNARE-mediated synaptic vesicle exocytosis. We discovered, through biochemical and structural analysis, that V100 is a target of Ca2+.calmodulin (Ca2+.CaM). Consequently, we performed in vivo studies in Drosophila that demonstrate the physiological significance of this interaction. CaM-binding deficient V100 fails to recruit CaM to synapses and is unable to rescue neuronal function during larval stages, but does not affect V100 protein stability, synaptic localization or neuronal development. To unravel the molecular mechanism of the CaM-dependent function of V100 in SNARE- mediated membrane fusion, we show that V100 can replace the v-SNARE synaptobrevin in the ternary v-/t- SNARE 'core'complex and form a remarkably similar V100/t-SNARE complex. Ca2+.CaM and t-SNAREs bind tightly to distinct sites on the N-terminal cytosolic region of V100. These findings have important ramifications for the sequence of molecular events leading to membrane fusion as well as its Ca2+-dependent regulation. In addition, loss of function of the V0 subunit "d" (V39) in fly neurons exhibits synaptic transmission defects indistinguishable from the loss of v-SNARE synaptobrevin or V100. Taken together, these findings serve as the framework of the overall goal to investigate the roles of the V-ATPase V0 subunits a and d in membrane fusion at the molecular and physiological levels and motivate the following three specific aims: Aim 1: To determine the structure-function relationships of the neuronal V0 subunits a1 and d. Aim 2: To investigate the binding specificity of CaM to neuronal subunit a1 and non-neuronal subunit homologues. The hypothesis is that Ca2+.CaM has a specialized regulatory function in neurons. Aim 3: To characterize in vivo the CaM- and SNARE-dependent functions of V0 subunit a1 and d at Drosophila synapses. When these studies are completed, we will have elucidated the roles of both subunits in V0 function in vesicle exocytosis. In particular, we will have established the molecular basis of the interactions of subunit a1 with calmodulin and SNARE proteins. By demonstrating that non-neuronal subunit a homologues are also targets of CaM, we will have uncovered additional regulatory role of CaM of other V0 subunit a fusion/secretion functions. Together, the structural, biochemical and in vivo studies encompassed in this proposal provide a rigorous test of an overdue integrative molecular model that reconciles v-ATPase V0 and Ca2+ Ca function with SNARE-mediated membrane fusion. PUBLIC HEALTH RELEVANCE: Synaptic vesicle exocytosis is a fundamentally important cellular process for nerve cells;its disruption or alteration can directly affect physiological activity of the brain, causing human disorders, such as neurodegenerative Alzheimer's and Parkinson diseases, as well as epilepsy, autism, schizophrenia, etc. The research proposed in this application will investigate, at the physiological and molecular level, the roles and regulations of the protein components, specifically subunits "a" and "d", of the V0 sector of the v-ATPase in exocytosis. Other disorders associated with defects in these components include type 1 diabetes mellitus due to mutations of subunit a and osteoporosis owing to mutations of subunit d.